Acute and chronic ethanol consumption differentially impact pathways limiting hepatic protein synthesis.

This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.

Glucocorticoids have a role in renal cortical expression of the SNAT3 glutamine transporter during chronic metabolic acidosis.

Glucocorticoids are involved in many aspects of regulation of acid-base homeostasis, including the stimulation of renal ammoniagenesis during chronic metabolic acidosis. Plasma glutamine is the principal substrate for ammoniagenesis under these conditions. Expression of the System N glutamine transporter SNAT3 is increased in the renal proximal tubules during acidosis. In vivo studies in rats using 1) sham and adrenalectomized rats, 2) the glucocorticoid receptor antagonist RU486, and 3) dexamethasone treatment demonstrated involvement of glucocorticoids in regulation of SNAT3 expression. Adrenalectomy attenuated the acidosis-induced increase in renal cortical SNAT3 mRNA approximately 40%, and treatment with dexamethasone (1 mg x kg(-1) x day(-1) sc) partially reversed this effect. RU486 also blunted the acidosis-induced increase in SNAT3 expression approximately 50%. Chronic dexamethasone treatment (0.1 mg x kg(-1) x day(-1) sc, 6 days) of normal rats slightly increased SNAT3 expression. In all cases, renal glutamine arteriovenous difference mirrored SNAT3 expression and activity in the proximal tubules, suggesting that SNAT3 regulates glutamine uptake during acidosis. These studies indicate that glucocorticoids regulate acid-base homeostasis during metabolic acidosis in part by regulating expression of the System N transporter SNAT3.

Stimulation of expression of the intestinal glutamine transporter ATB0 in tumor-bearing rats.

BACKGROUND: Glutamine supplementation ameliorates host catabolic response in tumor bearing states. The purpose of this in vivo study was to investigate intestinal glutamine transport and expression of glutamine transporter ATB(0) in methyl-cholanthrene (MCA)-sarcoma bearing rats. METHODS: Fisher-344 rats underwent subcutaneous flank implantation of MCA-sarcoma cells (saline as control) and were pair-fed an equal quantity of chow as controls, to account for tumor-induced anorexia, until tumors reached 10 or 20% body weight. Intestinal mucosal brush border membrane [3H]-Glutamine transport was measured. Glutamine transporter ATB(0) mRNA and protein levels were measured by real-time PCR and western blot techniques, respectively. RESULTS: Glutamine transport activity across the intestinal brush border membrane (BBM) was 3.7-fold higher in tumor-bearing rats (TBR) than in controls (TBR 153 +/- 22.6 vs. Control 41.9 +/- 9.7 pmol/mg protein/10s, P < .01). Transporter ATB(0) mRNA levels were 1.4-fold higher in tumor-bearing rats (Relative value TBR .61 +/- .12 vs. Control .43 +/- .1, P < .05). A 1.4-fold increase in transporter ATB(0) protein levels was observed in the tumor-bearing rats (Relative value TBR .52 +/- .07 vs. Control .37 +/- .04, P < .05). Circulating aortic plasma glutamine levels were 1.3-fold higher in tumor bearing rats ([Glutamine] = .63 +/- .02 Control vs. [Glutamine] = .74 +/- .01 mmol/l TBR, P < .0001). Portal venous plasma glutamine levels were also higher in tumor bearing rats ([Glutamine] = .47 +/- .01 Control vs. [Glutamine] = .60 +/- .02 mmol/l TBR, P < .0001). CONCLUSION: Intestinal brush border membrane glutamine transport activity, transporter ATB(0) mRNA and protein levels are up-regulate in tumor-bearing rats.

Branched-chain amino acid-enriched nutritional support in surgical and cancer patients.

Prolonged surgical stress and advanced malignant disease lead to systemic catabolism characterized by depletion of muscle protein and oxidation of skeletal muscle BCAA. BCAA oxidation provides energy for muscle and other organs and is the precursor for amino acid synthesis to replenish alanine and glutamine depleted in catabolic states. Persistent excessive catabolism leads to skeletal muscle wasting, negative nitrogen balance, and immune compromise. BCAAs, especially leucine, stimulate protein synthesis, inhibit proteolysis (in cell culture models and in animals), and promote glutamine synthesis. A number of small and diverse clinical trials studied the effects of BCAA-enriched nutritional support in moderately to severely stressed surgical and cancer patients. The findings of these clinical trials have been inconsistent; some show improved nitrogen balance, increased skeletal muscle protein synthesis, and reduced skeletal muscle catabolism whereas others show no significant improvement. The value of these trials is compromised by small sample size, heterogeneous patients, poor study design, varying degrees of metabolic stress, and inappropriate endpoints. More recent trials that evaluate clinical outcomes in hepatocellular carcinoma patients show promising results; in addition to improving metabolic parameters, BCAA-enriched oral supplementation improved morbidity and quality of life in patients undergoing major liver resection and chemo-embolization. In summary, the role of BCAAs in the nutritional support of stressed surgical and cancer patients remains to be clearly defined, despite their potential beneficial biological properties.

Regulation of amino acid arginine transport by lipopolysaccharide and nitric oxide in intestinal epithelial IEC-6 cells.

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 microg/ml) in serum-free Dulbecco's modified Eagles's medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0-500 micromol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-omega-nitro-L-arginine (NNA, 0-1000 micromol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectively. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 microg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 +/- 5; LPS: 92 +/- 20 pmol/mg/min, P < 0.05), with the System y(+) as the predominant arginine transport system, and a 2-fold increase of System y(+)CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y(+) maximal velocity (V(max), control: 1484 +/- 180; LPS: 2800 +/- 230 pmol/mg/min, P < 0.05) without affecting the transport affinity (K(m), control: 76 +/- 8; LPS: 84 +/- 14 micromol/L, p = NS). The LPS-induced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 +/- 6; LPS: 97 +/- 26 *; SNP: 22 +/- 0.4(+); LPS+SNP: 33 +/- 10.3(+) pmole/mg/min, *P < 0.01 and (+)p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 +/- 3.2; LPS: 59 +/- 2.7 *; NNA: 26.3 +/- 5.8; LPS + NNA: 127 +/- 18(+) pmol/mg/min; *P < 0.01 compared with control and (+)P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y(+) mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide.

Stimulation of intestinal glutamine absorption in chronic metabolic acidosis.

BACKGROUND: Amino acid glutamine plays a central role in inter-organ nitrogen transfer in acidosis, a compensatory mechanism that is essential in maintaining acidbase balance. Intestinal glutamine absorption is a key exogenous glutamine source in maintaining glutamine homeostasis. The purpose of this in vivo study was to investigate the regulation of intestinal glutamine absorption during chronic metabolic acidosis. METHODS: Metabolic acidosis in adult male Sprague-Dawley rats was induced by adding 1.5% NH4Cl to drinking water. [3H]-L-glutamine transport activity across brush border membrane vesicles and glutamine transporter ATB0 messenger RNA levels by relative reverse transcriptase-polymerase chain reaction were measured in rat jejunum. Data were analyzed by t test (P < .05). RESULTS: Acidosis occurred as early as 1 day and was partially compensated by 7 days. Glutamine transport in brush border membrane vesicles was increased after 2 days of acidosis. Chronic acidosis (7 days) resulted in an 8-fold increase of glutamine transport activity. The glutamine transport maximal capacity (Vmax) was stimulated 5-fold, while the transport affinity (Km) was not affected by acidosis. Relative reverse transcriptase-polymerase chain reaction showed a 2.5-fold increase of glutamine transporter ATB0 messenger RNA levels. CONCLUSIONS: Chronic metabolic acidosis stimulates intestinal glutamine absorption via a mechanism that involves an increase of functional membrane glutamine transporter units.

Insulin-like growth factor-2 activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

Insulin-like growth factor-2 (IGF-2) plays a pivotal role in regulating intestinal epithelial metabolism, growth, and proliferation, but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulatory mechanisms and intracellular signaling pathways involved in the regulation of IGF-2 on glutamine transport in cultured intestinal cells. Continuous incubation with IGF-2 stimulated glutamine transport activity in cultured IEC-6 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 48 hours) resulted in a 50% increase in transport activity (0.81+/-0.21 nmole/mg protein/min in IGF-2 cells vs. 0.57+/-0.15 nmole/mg protein/min in control cells) and a threefold increase in glutamine transporter ATB(0) mRNA levels. IGF-2 stimulated transport activity by increasing transport maximal capacity (V(max) 4.31+/-0.36 nmole/mg protein/min in IGF-2 cells vs. 2.51+/-0.23 nmole/mg protein/min in control cells) without affecting the transport affinity (K(m) 0.31+/-0.03 mmol/L glutamine in IGF-2 cells vs. 0.28+/-0.03 mmol/L glutamine in control cells). This IGF-2-induced glutamine transport activity was attenuated by actinomycin-D or cycloheximide. The levels of mitogen-activated protein kinases p42/44, MEK1/2, and p38 as well as protein kinase C levels were elevated in IGF-2-treated cells and inhibitors of mitogen-activated protein kinase MEK1 (PD 98059), mitogen-activated protein kinase p38, and protein kinase C (chelerythrine chloride) individually attenuated the IGF-2-induced glutamine transport. These data suggest that IGF-2 stimulates intestinal glutamine uptake in cultured rat intestinal epithelial cells via a mechanism that involves transcription and translation of the transporter. Activation of mitogen-activated protein kinases and protein kinase C cascades are involved in the regulation. This increase in glutamine uptake may occur to support intestinal cell growth and proliferation.

Metabolic acidosis stimulates intestinal glutamine absorption.

Glutamine is an essential nutrient for cell integrity during acidotic states such as shock, but the effect of extracellular pH on intestinal mucosal cell glutamine uptake is poorly understood. The purpose of this in vitro study was to investigate the intracellular signaling pathways involved in controlling intestinal glutamine transport during acidosis. Lowering the pH in the cell culture medium resulted in an increase in glutamine transport activity in a time- and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a twofold increase in glutamine transport activity (1.63+/-0.25 nmole/mg protein/minute in acidosis vs. 0.78+/-0.11 nmole/mg protein/minute in control) and a threefold increase in glutamine transport gene ATB(0) messenger RNA levels. This acidosis-induced increase in glutamine transport activity was due to a stimulation of transporter maximal transport capacity (V(max) 13.6+/-0.73 nmole/mg protein/minute in acidosis vs. 6.3+/-0.46 nmole/mg protein/minute in control) rather than a change in transporter affinity (K(m)=0.23+/-0.02 mmol/L glutamine in acidosis vs. 0.19+/-0.02 mmol/L glutamine in control). This acidosis-stimulated glutamine transport activity was blocked by actinomycin-D or cycloheximide. Cellular mitogen-activated protein kinase (MAPK) MEK1/2 and p42/44 levels were elevated in acidotic cells, and the acidosis-induced glutamine transport activity was blocked by the MAPK MEK 1 inhibitor PD 98059. Acidosis stimulates glutamine transport in Caco-2 cells via signaling pathways that lead to transcription of the glutamine transporter gene and translation of functional transporters. Mitogen-activated protein kinases are key intracellular regulators involved in this signal transduction cascade. An increased availability of glutamine to cells subjected to redox stress may help in maintaining cellular integrity.

Epidermal growth factor activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB(0) mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (V(max)) without altering the transporter apparent affinity (K(m)). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

Activation of intestinal arginine transport by protein kinase C is mediated by mitogen-activated protein kinases.

L-Arginine uptake by the small intestine can play a pivotal role in regulating nitric oxide synthesis and immune functions in catabolic states. We previously showed that protein kinase C (PKC) activation stimulates intestinal brush-border membrane arginine transport. However, the signaling pathways implicated in this activation have not been studied. The purpose of this study was to investigate the intracellular signal transduction pathways involved in the protein kinase C stimulation of arginine transport across the apical membrane of intestinal epithelial Caco-2 cells. [3H]-L-arginine transport activity, Northern blot analysis of mRNA levels of the intestinal arginine transporter CAT1, and Western blot analysis of the mitogen-activated protein (MAP) kinases phospho-p44/42 activity and phospho-MEK1/2 were measured in cultured Caco-2 cells treated with phorbol ester (phorbol 12-myristate 13-acetate, TPA; 0 to 0.5 micromol/L), and the MEK1 inhibitor PD 98059 (0 to 50 micromol/L). Phorbol ester stimulated intestinal arginine transport activity. Arginine transporter gene CAT1 mRNA, phospho-p44/42, and phospho-MEK1/2 levels were stimulated in phorbol ester-treated cells, compared with the control group. Phorbol ester stimulation of arginine transport activity and transporter CAT1 mRNA levels was blocked by PD 98059. These data suggest that phorbol ester stimulates arginine transport in Caco-2 cells via signaling pathways that lead to increased transcription and/or stabilization of CAT1 mRNA. Protein kinase C and MAP kinases MEK1/2 and p44/42 are key intracellular regulators involved in this signal transduction cascade.

Regulation of expression of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats.

During chronic metabolic acidosis, renal glutamine utilization increases markedly. We studied the expression of the system N1 (SN1) amino acid transporter in the kidney during chronic ammonium chloride acidosis in rats. Acidosis caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold increase in the cortex. Acidosis increased Na(+)-dependent glutamine uptake into basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 +/- 66 control vs. 651 +/- 180 pmol. mg(-1). min(-1) acidosis; BBMV, 1,112 +/- 189 control vs. 1,652 +/- 148 pmol. mg(-1). min(-1) acidosis, both P < 0.05). Na(+)-independent uptake was unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase in Na(+)-dependent glutamine uptake was eliminated by histidine, confirming transport by system N. SN1 protein was detected only in BLMV and BBMV from acidotic rats. After recovery from acidosis, SN1 mRNA and protein and Na(+)-dependent glutamine uptake activity rapidly returned to control levels. These data provide evidence that regulation of expression of the SN1 amino acid transporter is part of the renal homeostatic response to acid-base imbalance.

Protein kinase C activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

BACKGROUND: Glutamine is essential for the preservation of intestinal structure and function and its uptake by the bowel is augmented during catabolic states. However, the signal transduction pathways implicated in brush border glutamine transport have not been examined. The aim of this study was to investigate the intracellular signaling pathways involved in the regulation of accelerated intestinal glutamine transport. Our hypothesis was that the activation of intestinal glutamine transport involves protein kinase C (PKC) and is mediated by mitogen-activated protein kinases (MAPKs). METHODS: [3H]L-Glutamine (50 microM) transport activity and mRNA levels for the intestinal glutamine transporter ATB(0) were measured in intestinal epithelial Caco-2 cells. Confluent cells were treated with phorbol ester (PMA, 0-10 microM), the MAPK MEK inhibitor PD 98059 (0-100 microM), actinomycin (0-0.1 microM), MAPK p38 inhibitor SB 203580 (0-10 microM), protein kinase C inhibitor chelerythrine chloride (0-6.6 microM), or cycloheximide (0-10 microM) for 24 h. Data were analyzed by ANOVA with significance set at P < 0.05. RESULTS: Phorbol ester treatment increased intestinal System B glutamine transport activity by 75%, an increase that was blocked individually by PD 98059, chelerythrine chloride, actinomycin, and cycloheximide, but not SB 203580, an effect first noted at 6 h. The resulting activity increase was consistent with de novo synthesis of transporter units and enhanced expression of transporter gene ATB(0) as indicated by a threefold increase of ATB(0) mRNA levels in PMA-treated cells. CONCLUSIONS: Activation of glutamine transport in Caco-2 cells by phorbol ester occurs via signaling pathways that lead to transcription of the glutamine transporter gene. PKC and mitogen-activate protein kinase MEK are key intracellular mediators involved in this signal transduction cascade.

Specific reversible stimulation of system y(+) L-arginine transport activity in human intestinal cells.

L-Arginine, which is intimately involved in cellular immune functions and nitric oxide biology, is transported by intestinal cells largely via transport System y(+). The gut epithelium is exposed to various luminal amino acids at any given time, and therefore the purpose of this study was to study the regulation of luminal arginine transport by other amino acids. System y(+) L-arginine transport activity was measured in Caco-2 monolayers exposed to various amino acids. L-arginine and/or other System y(+) substrates specifically upregulated System y(+) transport activity twofold after 1 hour, with a response noted as early as 5 minutes. Non-System y(+) substrates did not affect L-arginine absorption. Kinetic analysis indicated that L-arginine exposure increased both System y(+) K(m) and V(max). Neither cycloheximide nor actinomycin affected this stimulation, indicating that the regulation did not involve transcription or translation. The System y(+) substrate activation effect was reversible. L-arginine transport activity returned to baseline within 3 hours when cells were reincubated in amino acid-free media. These data indicate that System y(+) arginine transport activity is rapidly and reversibly activated by System y(+) substrates via a mechanism consistent with transmembrane stimulation. These findings identify a mechanism by which luminal nutrients regulate arginine uptake by the gut.

Epidermal growth factor regulation of system L alanine transport in undifferentiated and differentiated intestinal Caco-2 cells.

Epidermal growth factor (EGF) in intestinal lumen regulates many gut epithelial cell functions. Influenced by growth factors at various differentiation stages, enterocytes execute the major task of absorbing nutrient amino acids. The purpose of this study was to investigate the effects of EGF on Na(+)-independent L-alanine transport in intestinal epithelial cells. Na(+)-independent [3H]-L-alanine transport was measured in the differentiating Caco-2 cells. In both the undifferentiated and differentiated states, L-alanine uptake occurred via a single saturable Na(+)-independent system L plus simple passive diffusion. System L activity decreased as the cells progressed from the undifferentiated to the differentiated state. Prolonged incubation with EGF (>30 hours) resulted in a 70% increase in system L activity in both undifferentiated and differentiated cells. EGF stimulated the system L V(max) without affecting K(m). System L activity stimulation was inhibited by chelerythrine chloride, cycloheximide, or actinomycin D. These data suggest that intestinal epithelial cell differentiation is associated with a decrease in system L transport capacity. EGF activates system L transport activity through a signaling mechanism involving protein kinase C, independent of cell differentiation state. Both cell differentiation and EGF regulation of system L activity occur via alteration of functional copies of the system L transporter.

Posttranslational alanine trans-stimulation of zwitterionic amino acid transport systems in human intestinal Caco-2 cells.

BACKGROUND: Neutral dietary amino acids, such as alanine, are transported across the gut lumen by both Na(+)-dependent (System B) and Na(+)-independent (System L) carriers, but the nature of the acute phase of substrate-induced uptake is unknown. This study examined the effects of acute amino acid substrate exposure on the rapid modulation of apical membrane alanine transport in cultured human intestinal cells. METHODS: System B and System L transport activity kinetics, as well as ATB(0) mRNA levels, were measured in confluent Caco-2 monolayers treated with various metabolic agents during short-term and extended time periods. RESULTS: Depleting the incubation medium of alanine attenuated both System B and System L uptake activities within 30 mins, with a complete return to baseline values within 3 h. Extracellular alanine added to depleted Caco-2 cells rapidly (within 5 min) increased alanine transport activities. Kinetic analysis showed that acute alanine exposure increased both K(m) and V(max) of each transport system, indicative of a trans-stimulation effect. Augmenting intracellular alanine levels using the cytosolic alanine aminotransferase inhibitor, aminooxyacetic acid, increased alanine uptake activity. Acute exposure to other substrates of Systems B and L also increased the uptake of alanine, while nonsubstrates did not affect alanine uptake. Cycloheximide or actinomycin did not affect substrate acute activation of System B, and the steady-state level of ATB(0) mRNA was not altered by amino acid exposure. CONCLUSION: Increasing alanine availability to intestinal cells, by either exogenous substrate exposure or inhibition of intracellular catabolism, acutely and reversibly increases apical membrane alanine transport activity via a posttranslation trans-stimulation mechanism.